Key Techniques for Somatic Embryogenesis and Plant Regeneration of Pinus koraiensis

: Korean pine is the dominant species of Korean pine forests. It is an economically valuable species that yields oil, high-quality timber and nuts, and it o ﬀ ers great prospects for further development. Complete regenerated plants of Korean pine were obtained via somatic embryogenesis using megagametophytes as the explant. The seeds of 27 families of Korean pine were collected to induce embryogenic lines. We compared the e ﬀ ects of explant collection time, family and medium components (concentrations of sucrose, plant growth regulators and acid-hydrolyzed casein) on embryogenic lines induction. The e ﬀ ects of plant growth regulators and L-glutamine contents on the proliferation and maturation of embryogenic cell lines were studied, and the germinating ability of di ﬀ erent cell lines was evaluated. The embryogenic lines induction percentage of Korean pine reached 33.33%. When 4.52 µ mol · L − 1 2,4-D and 2.2 µ mol · L − 1 6-BA were added to the medium of embryogenic lines proliferation, the ability of embryo maturation was the best (cell line 001#-100 was 135.71 · g − 1 fresh weight). Adding 1–1.5g L − 1 L-glutamine to the proliferation medium can improve the ability of embryo maturation (cell line 001#-100 was 165.63 · g − 1 fresh weight). The germination percentage of the three cell lines tested was signiﬁcant, and the highest was 66%. We report on successful regeneration and cryopreservation methods for somatic embryos of Korean pine. This technology could be used to propagate the excellent germplasm resources of Korean pine and to establish multi-varietal forestry. systems


Introduction
Somatic embryogenesis is the formation of embryo-like structures from somatic cells without gametic fusion [1,2]. Somatic embryos (SEs) are not only the best receptor system for genetic transformation [3,4], but are also invaluable for the large-scale propagation of excellent germplasm resources, and have many potential applications [5][6][7]. To date, somatic embryogenesis has been achieved for nearly 30 pine species. This method is used in production for some species, and has had remarkable economic benefits [8].  In July 2017, the best scheme selected in 2015 was used for EC induction culture (DCR + 35 g L −1 sucrose + 10.74 µmol·L −1 NAA + 6.66 µmol·L −1 6-BA + 0.8 mg L −1 CH + 6.5 g L −1 agar and 500 mg L −1 L-glutamine). Other culture methods were the same as in 2015. The sterilized fertilized megagametophytes were placed into Petri dishes, five explants per dish, and each family had 50 explants. The EC was cytologically observed. The fresh target EC was placed on a clean microslide, stained with 0.1% safranine staining solution, and then covered with a cover glass. The cover glass was tapped gently with the flat end of a pencil to spread the plant tissue evenly. The cells were observed and photographed immediately under an optical microscope (Olympus BX51 equipped with a Moticam 3000C camera). The EC of 001#-1, 001#-100 and 001#-34 were used as test materials. The proliferation medium was mLV [24] basic medium supplemented with 0.5 g L −1 CH, 30 g L −1 sucrose, 0.5 g L −1 L-glutamine and 4 g L −1 Gelrite (Phytagel™, Sigma-Aldrich, St. Louis, MO., USA). The hormone concentration was divided into three treatments: (1) 9.04 µmol·L −1 2,4-D + 4.4 µmol·L −1 6-BA; (2) 4.52 µmol·L −1 2,4-D + 2.2 µmol·L −1 6-BA; (3) 2.26 µmol·L −1 2,4-D + 0.44 µmol·L −1 6-BA. Embryonal mass (0.2 g) was transferred to the same composition in each subculture, each cell line with five replicates, subcultured every 2 weeks. The fresh weight of the embryogenic cell masses was measured after four subculture cycles, and the maturation ability of the embryogenic cell lines was measured under three different PGRs conditions. Proliferation experiment 2: Effect of L-glutamine on the proliferation of Korean Pine EC. The EC of 001#-1, 001#-100 and 001#-34 were used as test materials. The proliferation medium was mLV medium supplemented with 0.5 g L −1 CH, 30 g L −1 sucrose, 4.52 µmol·L −1 2,4-D, 2.2 µmol·L −1 6-BA and 4 g L −1 Gelrite. L-glutamine concentration was divided into three treatments (0.5, 1.0 and 1.5 g L −1 ). Embryonal mass (0.2 g) was transferred to the same composition in each subculture, each cell line with four replicates, and subcultured every 2 weeks. The fresh weights of the embryogenic cell masses were measured after four subculture cycles, and the maturation ability of the embryogenic cell lines was measured under three different L-glutamine conditions.

Maturation of SEs
The maturation ability of SEs was tested using the EC obtained from proliferation. The EC (100 mg) was transferred into a 50 mL centrifuge tube and the liquid proliferation medium without PGRs was added. Next, we shook the centrifuge tube violently to achieve full dispersal, and transferred the mixture to the filter paper with a pipette. A Brinell funnel was used for filtration, and then we put the filter paper with EC on the solid medium. Each cell line had between four and six replicates. The mLV medium contained 68 g L −1 sucrose, 75.66 µmol·L −1 abscisic acid (ABA), 500 g L −1 CH, 500 g L −1 L-glutamine and 10 g L −1 Gelrite. The number of SEs was recorded after being cultured at 23 ± 1 • C for 3 months.

SEs Germination and Plant Regeneration
In total, 50 mature SEs were randomly selected from three cell lines for the germination test. The SEs needed to be desiccation treated before germination, and the desiccation treatment was completed with 6-hole cell plate (Corning-Costar 3516), in which 3 holes were filled with two layers of dry sterile filter paper. We then put the SEs on the filter paper, and the remaining three holes were filled with sterile water. Finally, the cell plates were sealed with preservative film and cultured in the dark at 4 • C for 7 days. The cotyledon embryos were placed onto the germination medium. The germination medium was mLV medium supplemented with 2 g L −1 activated carbon, 20 g L −1 sucrose and 4 g L −1 Gelrite, cultured in the dark for 7 days, and then transferred to light culture (35 µmol·cm −2 ·s −1 light, 16 h light/8 h dark photoperiod, 23 ± 2 • C). The regeneration percentage of plantlets was recorded after 8 weeks of culturing.

Transplanting and Acclimatization of Regenerated Plants
After 16 weeks of culture, the rooted plantlets that developed from SEs were removed from the culture bottle with tweezers, the medium attached to the roots was washed off, and the plantlets were transplanted into a sterilized substrate (nutrient soil, vermiculite and perlite, at a volume ratio of 3:1:1). The plantlets were covered with plastic wrap to maintain high air humidity, and cultivated under the following conditions: 23 ± 2 • C, under a light intensity of about 35 µmol/(cm −2 ·s −1 ), and a 16 h light/8 h dark photoperiod. The plastic wrap was removed after 2 weeks, and the plants were watered regularly. The survival percentage of the plantlets was determined at 6 weeks after transplanting into the soil substrate.

Microscopy Observation
The paraffin section making method was performed as referred to by Li [25]. The cultures of different development stages were fixed in FAA fixative solution (formaldehyde: acetic acid:50% ethanol = 1:1:18). The fixed samples were dehydrated, soaked in wax and embedded in paraffin, stained with hematoxylin, then sectioned (the thickness of the section was 10 µm), and sealed with neutral balsam. The cells were observed under an optical microscope.

Data Statistics and Analysis Methods
The experimental data were processed using Excel 2003, and the average value and proliferation efficiency were calculated from these data. Single-factor analysis of variance (ANOVA) to evaluate the effects of PGRs, sucrose and CH was performed using SPSS 19 software (SPSS, Chicago, IL, USA).

Development of Explants in Different Periods
The development state of P. koraiensis seeds differed among the four collection times (Figure 1), ranging from the early embryo stage (Figure 1a  The red arrow points to the embryo head and is indicated by the letters 'eh', the yellow arrow points to the suspensor and is indicated by the letter s, the blue arrow points to the female gametophyte and is indicated by the letters 'fg', the yellow oval is the early embryogeny and is indicated by the letter e, and the red oval is the corrosion cavity and is indicated by the letter 'c'.

EC induction
The explants at the E1 stage were placed onto the induction medium, and cell proliferation was initiated at the micropylar end of the megagametophyte within 30 days of culturing (Figure 3a). The cytological observation indicated that there were vacuolated cells (Figure 3b), embryogenic cell The red arrow points to the embryo head and is indicated by the letters 'eh', the yellow arrow points to the suspensor and is indicated by the letter s, the blue arrow points to the female gametophyte and is indicated by the letters 'fg', the yellow oval is the early embryogeny and is indicated by the letter e, and the red oval is the corrosion cavity and is indicated by the letter 'c'.

EC induction
The explants at the E1 stage were placed onto the induction medium, and cell proliferation was initiated at the micropylar end of the megagametophyte within 30 days of culturing ( Figure 3a). The cytological observation indicated that there were vacuolated cells (Figure 3b), embryogenic cell

EC Induction
The explants at the E1 stage were placed onto the induction medium, and cell proliferation was initiated at the micropylar end of the megagametophyte within 30 days of culturing ( Figure 3a   The induction percentage of EC differed significantly among different families (p < 0.05). In 2015, the highest induction percentage was in family 135#, followed by family 108# and then 057# ( Figure  4). The average induction percentage of EC varied greatly among the different cone collection times. The EC induction percentage of E1 was the highest, followed by E2, E3 and then E4.  The induction percentage of EC differed significantly among different families (p < 0.05). In 2015, the highest induction percentage was in family 135#, followed by family 108# and then 057# (Figure 4). The average induction percentage of EC varied greatly among the different cone collection times. The EC induction percentage of E1 was the highest, followed by E2, E3 and then E4.  The induction percentage of EC differed significantly among different families (p < 0.05). In 2015, the highest induction percentage was in family 135#, followed by family 108# and then 057# ( Figure  4). The average induction percentage of EC varied greatly among the different cone collection times. The EC induction percentage of E1 was the highest, followed by E2, E3 and then E4.   Megagametophytes at the E1 stage from the 135# family were used as the explant. Among the media with combinations of NAA and 6-BA, the optimal medium for EC induction was DCR + 35 g L −1 sucrose + 10.74 µmol·L −1 NAA + 6.66 µmol·L −1 6-BA + 0.8 mg L −1 CH, with the highest induction percentage of 33.33% (Table 1). Among the media with combinations of 2,4-D + 6-BA, the optimal medium was DCR + 35 g L −1 sucrose + 27.14 µmol·L −1 2,4-D + 6.66 µmol·L −1 6-BA + 0.8 g L −1 CH, and the maximum induction percentage was 31.90% (Table 2). Across all media (combinations of NAA + 6-BA and 2,4-D + 6-BA), the factors could be ranked, from strongest influence on the induction percentage to weakest, as follows: NAA/2,4-D > sucrose > 6-BA > CH. In 2017, EC was produced from explants from 6 of the 24 families; the highest induction percentage was 12.00% and the lowest was 2.00% The induction percentage for other families was zero, and the induction percentage differed significantly among family sources ( Figure 5). Megagametophytes at the E1 stage from the 135# family were used as the explant. Among the media with combinations of NAA and 6-BA, the optimal medium for EC induction was DCR + 35 g L −1 sucrose + 10.74 μmol·L −1 NAA + 6.66 μmol·L −1 6-BA + 0.8 mg L −1 CH, with the highest induction percentage of 33.33% (Table 1). Among the media with combinations of 2,4-D + 6-BA, the optimal medium was DCR + 35 g L −1 sucrose + 27.14 μmol·L −1 2,4-D + 6.66 μmol·L −1 6-BA + 0.8 g L −1 CH, and the maximum induction percentage was 31.90% (Table 2). Across all media (combinations of NAA + 6-BA and 2,4-D + 6-BA), the factors could be ranked, from strongest influence on the induction percentage to weakest, as follows: NAA/2,4-D > sucrose > 6-BA > CH. In 2017, EC was produced from explants from 6 of the 24 families; the highest induction percentage was 12.00% and the lowest was 2.00% The induction percentage for other families was zero, and the induction percentage differed significantly among family sources ( Figure 5).

Proliferation Experiment 2: The Effect of L-Glutamine on the Proliferation and Maturation of Korean Pine EC
Different L-glutamine concentrations had no significant effect on the proliferation efficiency of EC (Table 5) and cell structure (Figure 7a,b), but had a significant effect on the development and maturation ability of SEs (p < 0.05) ( Table 6). When EC proliferated at 0.5 g L −1 L-glutamine, the number of late stage embryos in stage I was less than 0.5 g L −1 L-glutamine (Figure 7c,d), as was the number of mature embryos of stage III (cell line 001#-100 was the highest, which was 118.75·g −1 FW). In addition, when the concentration of L-glutamine was increased to 1 or 1.5 g L −1 , the development and maturation of the SEs of the three cell lines were improved, but the difference was not significant. When EC proliferated under the condition of 1.5 g L −1 L-glutamine, the number of late stage embryos of stage I was more than 0.5 g L −1 L-glutamine (Figure 7c,d, 001#-001 cell line had the best ability of embryo development and maturation). Furthermore, when under the condition of 1 g L −1 L-glutamine, 001#-100 and 001#-034 cell lines had the best capacities for SEs maturation (cell line 001#-100 was 165.63·g −1 FW, 001#-034 was 46.88·g −1 FW).    (Table 5) and cell structure (Figure 7a,b), but had a significant effect on the development and maturation ability of SEs (p ˂ 0.05) ( Table 6). When EC proliferated at 0.5 g L −1 L-glutamine, the number of late stage embryos in stage I was less than 0.5 g L −1 L-glutamine (Figure 7c,d), as was the number of mature embryos of stage Ⅲ (cell line 001#-100 was the highest, which was 118.75·g −1 FW). In addition, when the concentration of L-glutamine was increased to 1 or 1.5 g L −1 , the development and maturation of the SEs of the three cell lines were improved, but the difference was not significant. When EC proliferated under the condition of 1.5 g L −1 L-glutamine, the number of late stage embryos of stage I was more than 0.5 g L −1 L-glutamine (Figure 7c,d, 001#-001 cell line had the best ability of embryo development and maturation). Furthermore, when under the condition of 1 g L −1 Lglutamine, 001#-100 and 001#-034 cell lines had the best capacities for SEs maturation (cell line 001#-100 was 165.63·g −1 FW, 001#-034 was 46.88·g −1 FW).

Initiation of EC
The induction percentage of coniferous EC is usually very low, and depends heavily on the physiological stage of the explant, the location of the explant, the genotype of the families, the nutrient composition of the medium, the types and concentrations of growth regulators, and the culture conditions [23]. Our previous research shows that, as the age and physiological status of the mother tree increases, the callus induction percentage decreases significantly [19]. In most previous

Initiation of EC
The induction percentage of coniferous EC is usually very low, and depends heavily on the physiological stage of the explant, the location of the explant, the genotype of the families, the nutrient composition of the medium, the types and concentrations of growth regulators, and the culture conditions [23]. Our previous research shows that, as the age and physiological status of the mother tree increases, the callus induction percentage decreases significantly [19]. In most previous studies, EC has been induced from young tissues of coniferous species [26]. Many studies have reported that the EC induction percentages of Pinus and Picea are higher at the cleavage polyembryonic and prophase of cotyledon embryo stages than at other stages. In the present study, the best stage for the EC induction of P. koraiensis was the E1 stage (proembryo stage). The EC induction percentage was lower at the E3 and E4 stages than at the E1 stage, different from most other conifer species [23]. Our previous research shows that the highest induction percentage of EC was 1.67% [19]. In this study, the highest induction percentage was 33.33%, the lowest was 0.14%, and the induction percentage of EC was significantly increased. We found that there were significant differences in EC induction percentage among different families, like in our previous research [19]. The induction percentages of 24 families in 2017 were lower than those in 2015 (the highest induction percentage is 12%), and this may be related to the genotype of the families, or to the unsuitable development of immature seeds. Further verification is required in the later test process.
Phytohormones are the key substances controlling the process of somatic embryogenesis in culture in vitro. The induction and proliferation of the embryogenic cultures of many conifers proceed on standard nutrient medium supplemented with exogenous PGRs [27]. Auxin and cytokinin are used at the induction stage of Pinus [6]. In previous studies, the most commonly used auxin was 2,4-D, and the most commonly used cytokinin was 6-BA [28]. Auxin is considered to be the most critical factor in the induction stage [26]. In this study, combinations of NAA and 6-BA, or 2,4-D and 6-BA, at different concentrations were used to induce EC from explants of P. koraiensis. Both sets of combinations induced EC of P. koraiensis. Overall, the induction percentage was slightly higher with combinations of NAA and 6-BA than with combinations of 2,4-D and 6-BA, which was different from most other pine species [29][30][31].

Multiplication and Maturation of EC
EC is the foundation of regenerating plants on a large scale, and serves as an important material in genetic transformation. It is also an ideal system for studying the entirety of single-cell differentiation and the expression of totipotency [19]. The quality of EC not only affects the proliferation efficiency, but also affects the quantity and quality of SEs [32,33]. The quality of SEs is also a key factor in evaluating the success of somatic embryogenesis. In our study, the PGRs concentration significantly affected the EC proliferation and maturation, while L-glutamine concentration had no significant effect on the proliferation efficiency, but had a significant effect on the SEs' maturation. Therefore, during somatic embryogenesis, not only the proliferation efficiency but also the development and maturity ability of SEs should be considered.

Germination of Mature SEs
Among these phases, germination/conversion is regarded as the most important step in obtaining plantlets; this determines the success of this technique. Morphologically, mature conifer somatic embryos cannot germinate or convert into viable plantlets unless the embryos undergo partial desiccation treatment. This treatment has been used effectively to improve the germination/conversion of somatic embryos [34,35]. When SE was partially desiccated, the highest germination percentage was 66%. In the process of SEs germination, some SEs germinate abnormally [32,36,37], which limits the process of large-scale breeding. In this study, we found that the germinating ability of SEs of different genotypes was different. In the next step, we need to further optimize the key technology of EC proliferation of Korean pine, prolong the retention time of EC, enhance the ability of embryo maturation and germination, and lay the foundation for large-scale propagation.

Conclusions
In summary, this work demonstrates that efficient SE induction and establishment in continental and Mediterranean provenances of Korean pine depends not only on the mother tree but also on the developmental stage of the megagametophyte. Culture conditions during the EC proliferation stage significantly affect the maturation of SEs. Therefore, during somatic embryogenesis, not only the proliferation efficiency but also the development and maturity ability of SEs should be considered. We established systems for EC induction, somatic embryogenesis, plant regeneration and cryopreservation. These systems can be used to propagate the excellent germplasm resources of P. koraiensis, and for the establishment of multi-varietal forestry.
Author Contributions: L.Y. and H.S. conceived and designed the study. F.G. and C.P. collected plant materials and prepared SE samples for analysis. F.G. analyzed the results for experiments. L.Y., F.G. and H.W. wrote the paper. I.N.T. and A.M.N. revised the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgments: We thank two anonymous reviewers and the editor for comments that improved an earlier draft of this article.

Conflicts of Interest:
The authors declare no conflict of interest.